1. Field of the Invention
The present invention relates to cell lines established from insect tissues, especially relates to cell lines from L. xylina with high susceptibility to insect pathogenic microorganisms. These cell lines can be used in the mass production of insect pathogenic microorganisms and recombinant proteins.
2. The Prior Arts
The casuarina moth, Lymantria xylina Swinhoe (Order: Lepidoptera, Family: Lymantriidae, Genus: Lymantria), is one of the most damaging pests in the forest, mainly distributes in Taiwan, Japan, India, and the eastern coast of mainland China, which can eat all the leaves and twigs off a tree, stunt the growth and cause the death of plants. The toxic spines or hairs of L. xylina caterpillars provoke a cutaneous and immune reaction in human. Due to the extension of its host plant range and the increasing agricultural loss, this moth now becomes a significant quarantine pest.
The chemical insecticides are often used to kill the larvae of L. xylina to control the spread of L. xylina, which not only cause the problems of drug resistance and residual effects, but also involve in environmental contamination and resurgence of secondary insect pest species. Therefore, alternative control methods are studied to replace or to reduce the dependence of chemicals. Among them, using the insect pathogenic microorganisms to produce biopesticides is the most important method. Studies have revealed an lethal epizootic disease, nucleopolyhedrosis, of L. xylina larvae occurs from spring to early summer in Taiwan and mainland China, and the key pathogen was found to be L. xylina multiple nucleopolyhedrovirus (LyxyMNPV). LyxyMNPV is a rod-shaped virus and belongs to the baculovirus family. After infecting the host, the baculovirus buds budded virus (BV) to spread infection between cells inside the insect body and expresses the polyhedrin gene to produce polyhedrin. Multiple virions are found embedded in polyhedrin matrix in the nucleus known as occlusion bodies (OBs), which is also referred to as polyhedron inclusion bodies (PIBs), and the virus is therefore named as the nucleopolyhedrovirus (NPV).
The occlusion bodies can be released to outside from the lysed infected insects and further infect other insects to induce an epizootic disease. The spread of the disease is not only caused by horizontal transmission of NPV either from insect to insect or ingestion of the viral particles, but also be spread through vertical transmission from generations to generations. NPV are highly host-specific, have very species-specific tropisms among the invertebrates which cause no harm to humans, animals or other organisms. Studies have shown that the LyxyMNPV effectively suppress L. xylina population in field trials. Therefore the LyxyNPV can be used as a safe biological control agent in controlling the L. xylina populations. In addition, LyxyMNPV belongs to the baculovirus family, the latter is a core expression system of foreign protein production for medical and industrial applications, indicates the potential of being a recombinant protein expression system for LyxyMNPV.
Other than NPV, one of another main causes for epizootic disease is microsporidiosis inducing by microsporidia. The Nosema genus is the most common pathogen for microsporidiosis involved in the epizootic. Microsporidia propagate and infect host cells through the passages of meronts and spores. Since microsporidia play an important role in the regulation of insect populations, they have the potential to be used as biopesticides and be applied in pest control.
The insect pathogenic microorganisms can either be used as biopesticides in agriculture and forest pests, or be applied as a subject in studies of pathogenic genes and viral expression vectors for industrial or medical purposes. The priority is to have enough sources of the insect pathogenic microorganisms. The insect-infected baculovirus needs to be cultured in live cells, such as propagation through insect larvae or cell line culture. For example, multiplication of LyxyMNPV involved the large-scale rearing of L. xylina larvae. This process is labor intensive and several challenges need to be met (e.g. the conditions for breaking embryonic diapause the development of artificial diets, and the prevention of epizootic diseases) to obtain a reliable source of the host insect. In vitro production of LyxyMNPV in a highly susceptible insect cell line is an alternative solution. This strategy possesses advantages of: no contamination from other microorganisms during cultivation, screening and maintaining highly virulent LyxyMNPV strains.
Insect cell lines have the advantage for in vitro culture of pathogenic microorganisms. Hundreds of continuous cell lines have been established from over 100 insect species since Grace et al. established the first insect cell line in 1962 (Lynn, D. E., Development of insect cell lines: virus susceptibility and applicability to prawn cell culture. Methods in Cell Sci., 21(4):173-81.1999). These cell lines have been used broadly in researches of physiology, histology, embryology, molecular biology, pathology and insect virology. The baculovirus expression vectors and the production of recombinant proteins also need insect cell lines. However, there is no LyxyMNPV permissive cell line established so far. Besides, virus has specificity to host cell, each cell has different susceptibility to different virus. Therefore, establishment of proper insect cell line is critical for basic research and the following application.
In summary, the production of biopesticides can be enhanced to reach the safety and pest control purposes if a highly susceptible cell line to LyxyMNPV or other insect pathogenic microorganisms is established. The cell line can also be applied in the expression system of insect baculovirus to produce recombinant proteins for medical or research purposes.